Project Summary Worldwide, more than one million sexually transmitted infections (STIs) are acquired daily. Chlamydia trachomatis (C. trachomatis) and Neisseria gonorrhoeae (N. gonorrhoeae) are two such pathogens that show the greatest incidence of sexually transmitted disease (STD). If untreated, both chlamydia and gonorrhea can lead to serious health and reproductive issues, and yet, both are often asymptomatic in many infected individuals. The Centers of Disease Control and Prevention (CDC) and World Health Organization (WHO) strongly recommend routine screening of both men and woman. Several recent studies have also suggested the need for rapid point-of-care (POC) tests as most patients in the developed world are willing to wait only 20-30 minutes for results in the clinic. The advantages of a low cost, accurate, and true POC assay based on nucleic acid amplification for the detection of C. trachomatis and N. gonorrhoeae could facilitate early detection, thus potentially reducing transmission and sequelae, as well as improve therapeutic outcomes in the clinic. Beyond this setting, successful demonstration of such a rapid test could be expanded into the home-use market. RedVault Biosciences (RVB) has previously been awarded a Phase I grant (R43 CA200398-01A1) for its isothermal Target Reporter Construct (TRC) technology utilizing small RNAs, such as microRNAs. Here, RVB will develop a simple use, lateral flow (LF) test strip assay based on RVB’s proprietary TRC assay. Small RNAs (sRNAs) from C. trachomatis and N. gonorrhoeae will be used as targets with high specificity. By design, such sRNAs can be used as a primer of DNA replication in the TRC assay and not as the template traditionally used in nucleic acids amplification tests (NAATs). This novel utility of the target sample in a TRC assay simplifies library preparation and is well-suited for development of a POC test. To achieve these goals, three specific aims are proposed 1) determine the presence of bacterial sRNAs in clinical urine samples that have tested positive for C. trachomatis and N. gonorrhoeae, 2) develop a lateral flow test with RVB’s TRC technology, and 3) demonstrate feasibility with the test. Upon success with these goals, Phase II will include development of an integrated workflow that brings the steps of sRNA isolation, TRC CT/NG reaction, and LF detection into a simple, integrated POC device for the analysis of clinical samples.